Many proteins that have the potential to be useful human therapeutics have a xenogeneic origin. The use of xenogeneic proteins for therapeutic purposes may be advantageous for a variety of reasons, including, for example, the established success of hybridoma technology for raising antibodies in rodents, and the possibility of higher efficacy with a xenogeneic protein than with a human counterpart. Although xenogeneic proteins are a rich source of potential therapeutic molecules, they remain a relatively untapped one. One reason for this is that nonhuman proteins are often immunogenic when administered to humans, thereby greatly reducing their therapeutic utility. Additionally, even engineered proteins of human origin may become immunogenic due to changes in the protein sequence.
Immunogenicity is the result of a complex series of responses to a substance that is perceived as foreign, and may include production of neutralizing and non-neutralizing antibodies, formation of immune complexes, complement activation, mast cell activation, inflammation, hypersensitivity responses, and anaphylaxis. Several factors can contribute to protein immunogenicity, including but not limited to protein sequence, route and frequency of administration, and patient population. Immunogenicity may limit the efficacy and safety of a protein therapeutic in multiple ways. Efficacy can be reduced directly by the formation of neutralizing antibodies. Efficacy may also be reduced indirectly, as binding to either neutralizing or non-neutralizing antibodies typically leads to rapid clearance from serum. Severe side effects and even death may occur when an immune reaction is raised. One special class of side effects results when neutralizing antibodies cross-react with an endogenous protein and block its function.
Because of the clinical success of monoclonal antibodies, immunogenicity reduction of these proteins has been an intense area of investigation. Antibodies are a unique system for the development of immunogenicity reduction methods because of the large number of highly conserved antibody sequences and the wealth of high-resolution structural information. A number of strategies for reducing antibody immunogenicity have been developed. The central aim of all of these approaches has been the reduction of nonhuman, and correspondingly immunogenic content, while maintaining affinity for the antigen.
The dominant method in use for antibody immunogenicity reduction, referred to as “humanization”, relies principally on the grafting of “donor” (typically mouse or rat) complementarity determining regions (CDRs) onto “acceptor” (human) variable light chain (VL) and variable heavy chain (VH) frameworks (FRs) (Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA)). This strategy is referred to as “CDR grafting” (Winter U.S. Pat. No. 5,225,539). “Backmutation” of selected acceptor framework residues to the corresponding donor residues is often required to regain affinity that is lost in the initial grafted construct (U.S. Pat. No. 5,530,101; U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762; U.S. Pat. No. 6,180,370; U.S. Pat. No. 5,859,205; U.S. Pat. No. 5,821,337; U.S. Pat. No. 6,054,297; U.S. Pat. No. 6,407,213). Despite the significant clinical application of antibodies engineered using these methods, these methods remain nonrobust with regard to their ability to reduce immunogenicity. A number of humanized antibodies have elicited substantial immune reaction in clinical studies, with incidences of immune response as high as 63% of patients (Ritter et al., 2001, Cancer Research 61: 6851-6859).
The incomplete capacity of current humanization methods for immunogenicity reduction are due to significant limitations imposed by the donor-acceptor approach. Historically, the use of a single donor has been part of methods aimed at engineering a single xenogeneic antibody to be suitable as a human biotherapeutic. However, the use of a single acceptor is not required. On the contrary, the use of an acceptor antibody, and the use of global homology to select it, place substantial restrictions on the immunogenicity reduction process. A principal problem is that the use of overall sequence similarity between nonhuman and human sequences as a metric for human immunogenicity is fundamentally flawed. This means of measuring the degree of humanness does not accurately account for the underlying molecular mechanisms of immune response. The immune system does not recognize antigens on the basis of global sequence similarity to human proteins. Rather, immune cells, including antigen presenting cells (APCs), T cells, and B cells, recognize linear or conformational motifs comprising only a handful of residues. A key step in antigen recognition is the formation of peptide-MHC-T cell receptor complexes. APCs express MHC molecules that recognize short (approximately nine residue) linear peptide sequences, referred to as MHC agretopes. T cells express T cell receptors that recognize T cell epitopes in the context of peptide-MHC complexes. T cells that recognize MHC agretopes that are present in human proteins typically undergo apoptosis or become anergic, while T cells that recognize foreign agretopes bound to MHC molecules may participate in an immune response. Thus the relevant quantity for the immunogenicity of a protein is not its global sequence similarity to a human sequence, but rather its sequence content of individual human epitopes.
The donor-acceptor model and the use of global sequence homology that it imposes fails in practice. Because CDRs are treated as inviolable, structural incompatibilities are introduced at the CDR-FR boundaries. Grafting of foreign donor CDRs onto a human acceptor framework creates a substantial number of nonhuman epitopes in each variable chain, including not only the epitopes in the foreign CDRs, but also the large number of epitopes at the FR-CDR boundaries. This FR-CDR incompatibility is evident when one backs away from global homology and looks at more local sequence homologies. CDR grafting generally maximizes the donor-acceptor homology of the frameworks at the expense of the CDRs (Clark, 2000, Immunology Today 21: 397-402). Ironically this frequently results in lower global homology to human antibodies. In reality, the “cut and paste” approach to imparting the functional determinants of a nonhuman antibody onto the framework of a human one is unnecessary, as careful analysis of the antigen binding determinants of antibodies shows that, in fact, the majority of CDR residues are not involved in binding antigen (MacCallum et al., 1996, J. Mol. Biol. 262: 732-745). FR-CDR incompatibility causes not only immunological problems at the sequence level, but also causes conformational problems at the structural level. As a result, humanization methods based on CDR grafting often result in antigen affinity losses of 10-100-fold, necessitating backmutation to donor residues within the framework. This process of backmutation is a hallmark of essentially all current humanization efforts, and because it introduces yet additional nonhuman epitopes, highlights the inefficiency of these methods.
Methods that take an immune epitope approach to reducing antibody immunogenicity have been explored (U.S. Pat. No. 5,712,120; US 2003/0153043). Central to these methods is the determination of sequences within a xenogeneic antibody that are in fact immunogenic epitopes. Different methods for determination of immunogenicity both theoretical and experimental have been described and include determination of potential for amphipathic helix formation, binding to MHC, reactivity in a T-cell activation assay. A distinguishing feature between these strategies and the present invention is that the present invention makes no presumption as to the immunogenicity of specific epitopes. Rather, the primary goal is to maximize the content of human linear sequence strings in the xenogeneic antibody as determined by comparison to an alignment of human sequences. The relevant sequence dataset comprises strings that are nonimmunogenic for all relevant reasons, including lack of interaction with MHC, lack of interaction with T cell receptor, lack of proper processing necessary for presentation, and tolerance.
It is noted that the methods described in U.S. Pat. No. 5,712,120 and US 2003/0153043 suffer additionally in that they fail to address a significant concern for local level sequence engineering, namely the requirement for maintaining protein structure, stability, solubility, and function. Thus, although the sequence string approach to immunogenicity reduction is more accurate than CDR grafting, it will be optimal when coupled with protein design methodology that takes into account both local sequence content and conformational compatibility at the local and global structural level. In addition to providing scoring functions for assessing host string content, the present invention also describes scoring functions that evaluate other relevant properties of a protein that may be employed for the simultaneous immunogenicity reduction and structural and functional optimization of proteins.
In summary, the donor-acceptor model imposes significant restrictions on the immunogenicity reduction process. With regard to sequence, global sequence homology is an inappropriate metric for immunogenicity. With regard to structure, backmutations are needed to repair conformational incompatibilities, thereby creating or reintroducing nonhuman epitopes. The present invention describes a novel method for antibody immunogenicity reduction that steps outside of the donor-acceptor model, and thus the sequence and structural restrictions it imposes. The central strategy of the described method is that it maximizes the content of human linear sequence strings. In this way immunogenicity is addressed at the local sequence level, typically by utilizing the local sequence information contained in an alignment of human sequences. This strategy not only provides a more accurate measure of the immunogenicity, it enables substitutions to be designed in a forward rather than backward manner to repair problems introduced by the graft. In effect, by addressing immunogenicity at the local sequence string level, the optimal balance between binding determinants and humanness can be designed.
The present invention describes a novel method for reducing the immunogenicity of proteins that leverages the nonimmunogenic information contained in natural human sequences to score protein sequences for immunogenic content at the sequence string level. Furthermore, the described method capitalizes on recent advances in computational sequence and structure-based protein engineering methods to quantitatively and systematically determine the optimal balance between human sequence content and protein functionality. Because of the wealth of human sequence information available for the immunoglobulin protein family, application to human antibodies is emphasized. Applications to other proteins are also possible.